FTIR Study of Bacteriorhodopsin

细菌视紫红质（bR）是一种光驱动质子泵，其功能包括两个关键的基于膜的过程：主动转运和能量转导。尽管对bR和其他膜蛋白进行了广泛的研究，但这些过程的分子基础尚不清楚。罗斯柴尔德博士的实验室开发了一种基于傅里叶变换红外（FTIR）差分光谱的方法，可以在单个蛋白质残基的水平上研究这些过程。这项工作提供了bR光循环过程中特定残基质子化状态和环境变化的信息。这项研究的目标将是利用这种方法来开发bR泵机制的越来越详细的图片。时间分辨FTIR差分光谱、偏振FTIR和衰减全反射等最新改进的方法将促进这些研究。与H.G. Khorana博士的团队合作，开发了基于位点定向诱变的新方法，用于将FTIR差谱中的波段分配给单个蛋白质残基的振动。这些技术允许研究bR质子运输途径中Asp基团和其他残基的质子化变化序列；改变了膜中单个COOH基团的bR -螺旋方向和pK。平行研究将使用时间分辨紫外/可见光谱和共振拉曼光谱进行。FTIR研究还将继续对来自halobium halobium的两种相关膜蛋白进行研究：halorhodopsin (hR)，一种光驱动的氯泵和感觉视紫红质（sR-I），一种趋光性光受体。

Bacteriorhodopsin (bR) is a light driven proton pump whose function includes two key membrane based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, the molecular basis of these processes is unknown. Dr. Rothschild's laboratory has developed an approach based on Fourier transform infrared (FTIR) difference spectroscopy which allows these processes to be investigated at the level of single protein residues. This work is providing information about changes in the protonation state and environment of specific residues during the bR photocycle. The goal of this research will be to utilize this approach to develop an increasingly detailed picture of the bR pump mechanism. These studies will be facilitated by recent improved methods developed for time-resolved FTIR difference spectroscopy, polarized FTIR and attenuated total reflection. In collaboration with Dr. H.G. Khorana's group new methods have been developed, based on site- directed mutagenesis, for assigning bands in FTIR difference spectrum to the vibrations of individual protein residues. These techniques allow the investigation of the sequence of protonation changes of Asp groups and other residues in the bR proton transport pathway; changes in the orientation of the bR alpha- helices and changes in pK of individual COOH groups in the membrane. Parallel studies will be conducted using time-resolved UV/visible spectroscopy and resonance Raman spectroscopy. FTIR studies will also be continued on two related membrane proteins from Halobacterium halobium: halorhodopsin (hR), a light driven chloride pump and sensory rhodopsin (sR-I), a phototaxis light receptor.